A photoactivable phospholipid analog that specifically labels membrane cytoskeletal proteins of intact erythrocytes

Abstract

A radioactive photoactivable analogue of phosphatidylethanolamine, 2-(2-azido-4-nitro-benzoyl)-1-acyl-sn-glycero-3-phospho[14C]ethanolamine ([14C]AzPE), was synthesized. Upon incubation with erythrocytes in the dark, about 90% of [14C]AzPE spontaneously incorporated into the cells; of this fraction, about 90% associated with the membrane, all of it noncovalently. Upon photoactivation, 3-4% of the membrane-associated probe was incorporated into protein. Analysis of this fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as well as extraction of labeled membranes with alkali or detergent, showed that the probe preferentially labeled cytoskeletal proteins. [14C]AzPE appears to be a useful tool for the study of lipid-protein interactions at the cytoplasmic face of the plasma membrane of intact cells.

Calcium induces transbilayer redistribution of all major phospholipids in human erythrocytes

“…The distribution of phospholipids is normally asymmetric across the plane of the plasma membrane of erythrocytes (Verkleij et al, 1973;Kahlenberg et al, 1974;Gordesky et al, 1975) and platelets (Schick et al, 1976;Chap et al, 1977;Bevers et al, 1982), with the aminophospholipids phosphatidylethanolamine (PE)1 and PS mostly, or completely (respectively), concentrated in the inner leaflet and the neutral phospholipids PC and Sph mostly, or completely (respectively), concentrated in the external leaflet. Studies of the transbilayer movement of specific phospholipids (Seigneuret & Devaux, 1984;Daleke & Huestis, 1985; Zachowski et al, 1986;Tilley et al, 1986; Connor & Schroit, 1987; Morrot et al, 1989) and their interaction with cytoskeletal proteins (Pradhan et al, 1989(Pradhan et al, , 1991 have implicated an ATP-dependent aminophospholipid translocase in the maintenance of this asymmetric lipid distribution. This enzyme moves PS, and less efficiently PE, from the outer to the inner leaflet in an energy-dependent reaction.…”

Orientation of the benzophenone group at various depths in bilayers

“…It is quite possible that such shallow probes may only label proteins near the membrane surface, and it is not surprising that a recent report on a similar phosphatidyl ethanolamine-based azide probe indicated selective labeling of spectrin in erythrocytes. 23 The present work indicates that benzophenone-based phospholipids can be conveniently prepared and can give rise to fairly high cross-linking yields, i.e., 35-40%. In conclusion, the results obtained here using 1-3 indicate a sharper range of carbon functionalization and highlight the advantages associated with use of phospholipids with a fatty acyl chain bearing a hydrophobic tail. A tail longer than 4-6 carbon atoms can be utilized so that it interdigitates24 with the opposite bilayer, thus providing a more specific orientation of the photoactive group.…”

Functional involvement of proteins, interacting with sphingolipids, in sphingolipid transport to the canalicular membrane in the human hepatocytic cell line, HepG2?

“…By photoactivation, adjacent proteins potentially involved in lipid processing and dynamics, including sorting, can thus, in principle, be identified. 17,[26][27][28][29][30] Before photolabeling experiments, the metabolism of the photoreactive Cer analog in HepG2 cells was studied. During these experiments, photoactivation of the photolabel was avoided by performing the experiments under subdued or red light.…”